Throughout this application, various publications are referenced by arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed in this application.
Gangliosides are prominent cell-membrane components of melanoma and other tumors of neuroectodermal origin. Many different gangliosides have been described in melanoma cells. These include GM3, GM2, GMI, GD3, GD1A, GD1B, GD2, GT1B, 9-0 acetyl GD3, and GT3 (1-2). Four of these, GM2, GD2, GD3, and 9-0 acetyl GD3, have attracted special attention because of wide distribution on melanoma cells and limited distribution on cells outside the central nervous system (3-6). The importance of GD2 and GD3 has been suggested by regression of melanoma and neuroblastoma metastases in some patients treated with anti-GD3 and anti-GD2 monoclonal antibodies (MmAb) (7-9). Many patients have received vaccines containing melanoma cells expressing these gangliosides (10-13). These studies have shown that GM2 antibodies were frequently induced, antibodies against GD2 were occasionally induced, and antibodies against GD3 and 9-0 acetyl GD3 were never detected. Due to uncertainties about expression of each ganglioside in the vaccines, or on the target cells used for serological analysis, the relative immunogenicity of GM2, GD2, GD3 and 9-0 acetyl GD3 remained unknown.
In attempts to induce active immunity against melanoma, we focused on GM2 in initial studies because its distribution on cell lines (defined by anti-GM2 MmAb 5.3) and on various tissues (detected by extraction and thin layer chromatography) was quite restricted (3). In a series of experiments in the mouse, we have identified immunizing procedures that facilitate the serologic response to GM2 and other gangliosides (14-15): pretreatment with low dose cyclophosphamide and immunization with GM2 attached to adjuvant-carriers such as BCG or Salmonella minnesota mutant R595. Trials comparing these approaches in early stage melanoma patients demonstrated BCG to be a significantly better adjuvant than R595 and patients pretreated with a low dose of cyclophosphamide had significantly higher titers of anti-GM2 antibody than those not receiving this pretreatment (12). IgM antibodies were induced in 72% of patients receiving the BCG-GM2 vaccine, and these were capable of lysing human tumor cells in the presence of human complement. IgG antibodies were detected in 25% of the immunized patients. The pattern of primary and secondary antibody response to immunization was most consistent with GM2 acting as a T cell independent antigen.
In the study reported here, we apply the same immunization approach, CY +BCG - purified ganglioside, to test the immunogencity of GD2, GD3, and 9-0-acetyl GD3. The relative immunogenicity of these gangliosides was examined in AJCC stage III and IV melanoma patients who were free of detectable disease after surgery. 9-0-acetyl GD3 is identified as a second effective immunogen.
Nothing yet is known about the immunogenicity of 0-acetyl-GD3. Although structually close to GD3, the molecule is antigenically different (5,6 16-17). While Ravindranaths et al. have recently described 2 melanoma patients with antib-ody reactivity against 9-0-acetyl and/or 4-0 acetyl GD3, the incidence of such antibodies in melanoma patients was unknown (18) . In this study we report our attempts at chemically synthesizing 0-acetyl-GD3 and characterizing the properties of the products obtained. As immunization of patients with malignant melanoma with these derivatives is envisaged, we examined their immunogenicity in the mouse, by analysing the humoral immune response after vaccination with 0-acetyl-GD3 derivatives.